The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary.

The human oocyte appears to be particularly prone to meiotic errors, and the incidence of these errors is strongly influenced by maternal age. We have initiated studies of human oocytes from unstimulated ovaries and have observed age-related effects on the meiotic process in oocytes from unselected antral follicles. Specifically, in oocytes obtained from donors over the age of 35 years, the majority of oocytes that extruded a first polar body in culture and arrested at second meiotic metaphase had aberrations in spindle formation and chromosome alignment. Similarly, observations of a limited number of oocytes at first meiotic metaphase suggest disturbances at this stage of meiosis as well. Finally, preliminary results of non-disjunction studies suggest that the frequency of errors in chromosome segregation at the first meiotic division is influenced by donor age in in-vitro matured oocytes as it is in oocytes undergoing meiotic maturation in vivo. These data provide direct evidence that the meiotic competence of oocytes from unstimulated ovaries declines with donor age. Similarly, studies of in-vitro fertilization (IVF) pregnancies in older women indicate that the developmental competence of the human oocyte declines with age. Since both meiotic and developmental competence are acquired during the late stages of oocyte growth, we postulate that an age-related decline in the process of folliculogenesis results in reduced oocyte quality and that the well characterized age-related increase in meiotic non-disjunction is one symptom of compromised oocyte growth.

Limiting multiple pregnancy in in vitro fertilization/embryo transfer (IVF-ET) cycles.

PURPOSE: Our purpose was to assess how the number of embryos transferred can be adjusted to limit multiple gestations. METHODS: A retrospective analysis of 535 consecutive embryo transfers for the years 1991-1993 was conducted. RESULTS: Fewer than three embryos were associated with a low pregnancy rate. Pregnancy rates were highest in women less than 35 when four or more embryos were transferred. With four or more embryos, multiple gestation pregnancy correlated with the number of high-quality embryos transferred. The risk of triplets and quadruplets was greatest for women less than 40. CONCLUSIONS: Multiple-embryo transfer carries a risk of plural gestation. The risk of multiple pregnancy cannot be eliminated without decreasing the pregnancy rate. The risk of high-order multiple pregnancy was best correlated with the number of good-quality embryos transferred. While all are at risk, patients younger than 40 were at highest risk.

The effect of Y-chromosome alpha-satellite array length on the rate of sex chromosome disomy in human sperm.

Trisomy is the leading known cause of mental retardation and pregnancy loss in humans, yet virtually nothing is known of the underlying nondisjunctional mechanisms. Since studies of other organisms suggest an association between centromere size or sequence and meiotic nondisjunction, we recently initiated studies to examine the effect of centromere size variation on human nondisjunction. In the present report, we summarize studies correlating variation in the size of the Y-chromosome centromere with sex chromosome nondisjunction. In one set of studies, we used pulsed-field gel electrophoresis to estimate Y-chromosome alpha-satellite array lengths in normal males, and correlated these values with Y-chromosome sperm disomy levels as determined by fluorescence in situ hybridization. In a second set of studies, we determined the Y-chromosome alpha-satellite array length of 47,XYY males, since the karyotypes of these individuals are a consequence of Y chromosome nondisjunction. Neither set of studies provided evidence for an effect of Y-chromosome alpha-satellite array length on Y-chromosome nondisjunction. Thus, if there is an association between Y-chromosome centromere size and nondisjunction, the effect is subtle and below the detection levels of the present study or involves extreme size variants that were not represented in the present study population.

Clinical experience with synthetic serum substitute as a protein supplement in IVF culture media: a retrospective study.

OBJECTIVE: Our objective was to evaluate the efficacy of Synthetic Serum Substitute (Irvine Scientific--Materials Section, Santa Ana, CA), a globulin-enriched protein preparation containing human serum albumin for supplementation of IVF culture media. DESIGN: We retrospectively analyzed IVF cycles performed at MacDonald Womens Hospital between January 1992 and November 1994. IVF cycles were reviewed and classified according to the nature of protein supplementation used in the embryo culture medium. Three protein supplements utilized during this time period were compared: Synthetic Serum Substitute (SSS), Plasmanate (PL), and maternal serum (MS). RESULTS: Although clinical pregnancy rates among the three treatment groups were not statistically different, there was a definite trend toward a higher pregnancy rate with SSS supplementation (SSS, 38.2%; MS, 28.0%; and PL, 24.9%). Embryos grown in SSS-supplemented culture media had a significantly higher implantation rate (17.8 vs 10.4 and 10.3%, respectively, for MS and PL). Preliminary data also suggest that human embryo development and blastulation in vitro were enhanced by this protein supplement. CONCLUSIONS: The higher implantation rate with SSS suggests that it may be superior to both maternal serum and Plasmanate in supporting human embryo development in vitro. Whether blastocysts derived from PL- and SSS-supplemented media are able to implant and give rise to clinical pregnancies remains to be seen.

Non-disjunction in human sperm: evidence for an effect of increasing paternal age.

In humans, the relationship between advancing maternal age and the incidence of trisomy has been long established, but the possible effect of increasing age of the father remains controversial. Using a fluorescence in situ hybridization (FISH) approach to directly examine individual sperm for aneuploidy of the sex chromosomes and chromosome 18, we have analyzed approximately 400,000 sperm from 24 men aged 18-60 years. There was no obvious relationship between increasing age and disomy 18, but the incidence of XY,YY and XX disomy all were significantly elevated among older men. This suggests that older men, like older women, have an increased likelihood of producing aneuploid offspring by comparison with their younger counterparts.

Analysis of chromosome behavior in intact mammalian oocytes: monitoring the segregation of a univalent chromosome during female meiosis.

To monitor the behavior of specific chromosomes at various stages of mammalian female meiosis, we have combined immunofluorescence staining and fluorescence in situ hybridization (FISH) on intact oocytes. We have utilized this technique to evaluate the behavior of the single X chromosome in oocytes from XO female mice, providing the first observations on segregation of an achiasmate chromosome during mammalian female meiosis and its effect on the meiotic process. As has been described in other species, we found that the univalent chromosome could either segregate as an intact chromosome to one pole or divide equationally at the first meiotic division. Our results also indicate that the presence of a univalent chromosome causes severe meiotic disruption during mammalian meiosis, affecting the alignment and segregation of other chromosomes in the complement. Despite these meiotic abnormalities, the vast majority of oocytes from XO females were able to resume and successfully complete the first meiotic division. This is in contrast to previous studies of male mice with sex chromosome abnormalities where the presence of a univalent acts to arrest meiosis at metaphase of the first meiotic division. This sex-specific difference in the ability of a cell with a univalent chromosome to initiate anaphase suggests that cell cycle control differs between male and female meiosis and that monitoring of meiotic chromosome behavior is less efficient in the female. The combined use of immunofluorescence staining and FISH on intact oocytes has obvious application to the study of meiotic chromosome non-disjunction in the human female. Simultaneous study of the meiotic cell cycle, protein components of the meiotic apparatus, and chromosome-specific behaviors during mammalian female meiosis provides a new approach to defining age-related changes in the meiotic process that result in increased chromosome malsegregation.

Sex hormone modulation of flow velocity in the parametrial artery of the pregnant rat.

The objective of the study was to correlate the disparate distal/proximal state of maturation of uterine segments in the rat with the hemodynamics of the parametrial uterine artery. Flow velocity was determined by the methylene blue photometric analysis. In nonpregnant animals, the velocity in the distal (VD-->P) and proximal (VP-->D) portions of the parametrial artery were similar, but in late pregnancy, VD-->P was higher than VP-->D. The difference was associated with an increased width of the distal part of the parametrial artery. In addition, the distance from the proximal end of the parametrial artery where the D-->P and P-->D streams collided decreased in animals in late pregnancy, compared with nonpregnant animals. Treatment with estradiol and RU-486 reversed the pregnancy-related hemodynamic changes. The pregnancy-related shift in flow velocity in the parametrial artery is crucial to rat fetal survival in utero and may be a mechanism in vivo for promoting maturation of distal, compared with proximal, segments of the uterine horn.

Distribution of unesterified cholesterol-containing particles in human atherosclerotic lesions.

The objective of the study was to characterize unesterified cholesterol particles in human aorta and to correlate the findings with the severity of aortic atherosclerosis. Human tissues were processed under conditions that preserve deposits of unesterified cholesterol agglomerates. Filipinfluorescence was determined by using a novel triple band pass filter. The pattern of unesterified cholesterol deposits was age related and correlated with the severity of atherosclerosis. We found three types of deposits: 1), small spherulites (3 to 5 mu), which were depicted in both the media and intima in individuals as early as age 16, and which, in more advanced ages, showed an increase in density and a tendency to aggregate extracellularly throughout the intima in clusters; 2), elongated structures (10 to 30 mu in the middle zone of the intima), the density of which was directly related to the severity of atherosclerosis; and 3), large (100 mu), irregular deposits found mainly in the core of atherosclerotic plaques. The medium size deposits, compared with those found in the core of atherosclerotic plaques, retain their overall size (10 to 30 mu), uniformity (oval elongated), and localization (middle zone of the intima). On the basis of these observations we hypothesize cholesterol deposition in two stages of aggregation: 1), early degradation of infiltrating low density lipoprotein particles forming unesterified cholesterol-rich vesicles in the vessel wall, followed by aggregation to spherulites in the lower part of the intima; and 2), more massive agglomeration of particles containing unesterified cholesterol and calcium phosphate in the midzone of the intima. Because in the second stage of aggregation the transition of cholesterol to the solid state has already occurred, it is irreversible.

Expression of the insulin-like and platelet-derived growth factor genes in human uterine tissues.

The human uterus repeatedly exhibits cyclic biochemical and cytological changes during the reproductive period of life. These changes are the result of a well-characterized endocrine network involving the hypothalamus, pituitary, and ovary. The exact nature of the mechanism(s) by which the sex steroids act on the uterus remains to be elucidated. Possible local mediators of hormonal action on the uterus include polypeptide growth factors. Using the method of RNA transfer blot hybridization, we have analyzed tissue samples from the cycling human endometrium and tissue samples of human myometrium and myometrial benign tumor (leiomyoma) for the presence of platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) RNA. All the uterine tissues examined possessed RNA for PDGF-B chain and IGF-I and -II. Two transcripts were observed for PDGF-B chain, four were observed for IGF-I, and eight were observed for IGF-II. Overall, the relative abundance of PDGF-B chain RNA was consistent in all of the uterine tissues examined. In contrast, IGF RNA relative abundance varied. IGF-I RNA was highest in late proliferative stage endometrium, and IGF-II RNA was highest in early proliferative stage endometrium. Both IGF-I and IGF-II RNAs were greater in amount of leiomyoma than in myometrium. The increased IGF-I RNA in late proliferative-stage human endometrium correlates with the known elevation of estradiol secretion by the ovary and the increased concentration of uterine estradiol receptors during this stage of the menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Myometrial oxytocin receptors levels in the pregnant rat are higher in distal than in proximal portions of the horn and correlate with disparate oxytocin responsive myometrial contractility in these segments.

Distal (D) segments of the pregnant rat uterine horn express myometrial oxytocin receptors (MORs) earlier than proximal (P) segments (day 18 vs. 20, respectively); the levels in D segments remain higher than in P segments throughout days 21-22 and correlate with the segment-specific myometrial sensitivity to oxytocin. While progesterone (P4) had no effect on MOR levels, RU486 increased (t1/2 6-12 h) MOR levels both in D and P segments, particularly in days 12-17, and the levels in the P segment equaled those in the D segment. Estradiol had no effect on MOR levels in days 20-22; in days 16-19 estradiol increased MOR levels particularly in the P segment, and the levels in the latter were higher than in the D segment. Capillary plasma P4 levels were higher in P vs. in D myometrial segments. These results indicate, in the pregnant rat, a local uterine control of D greater than P MOR expression by P4 withdrawal beginning in day 18. We hypothesize that the D greater than P MOR expression determines a role for oxytocin in initiating myometrial contractions in the D segment, while in active labor another class of agent(s) assume that function in more proximal segments.

Cultured human ectocervical epithelial cell differentiation is regulated by the combined direct actions of sex steroids, glucocorticoids, and retinoids.

During the menstrual cycle, the ectocervical epithelium undergoes a cyclic change in differentiation that is correlated with changes in the circulating levels of estrogens and progestins. To better understand the mechanisms underlying this regulation, we have developed an ectocervical epithelial cell (ECE cell) culture system that responds in a physiological manner to stimulation by estrogens and progestins. We have recently proposed that four classes of hormones (retinoids, estrogens, progestins, and glucocorticoids) may play an important role in regulating differentiation. In the present paper we test this hypothesis directly by making detailed dose-response curves. Our results demonstrate that estrogen increases ECE cell differentiation, as measured by release of cornified envelopes (superficial cells), while progesterone decreases envelope production. The effects are dose dependent and within the expected physiological range (0.01-10 nM estrogen; 0.1-100 nM progesterone). Very low concentrations of Ro 13-6298 (0.01-10 nM), a synthetic retinoid, decreased envelope production, while hydrocortisone (0.1-50 nM) increased envelope production and cell growth. Importantly, agents that enhance envelope production are neutralized by agents that reduce envelope formation and vice versa. Based on these findings we conclude 1) that the differentiation-enhancing actions of glucocorticoids and estrogens can be antagonized by either progestins or retinoids, and 2) that glucocorticoids and retinoids are likely to determine the ECE cell differentiation set-point in vivo, with the sex steroids directly modulating the phenotype of the ECE cells around this set-point during the menstrual cycle. Moreover, these results appear to explain some of the clinical descriptions of changes in ectocervical cell morphology resulting from hyper- and hypoestrogenic stimulation.

Retinoids, sex steroids and glucocorticoids regulate ectocervical cell envelope formation but not the level of the envelope precursor, involucrin.

In spite of extensive study of the reproductive tract, little knowledge is available regarding the function of ectocervical epithelial (ECE) cells. In the present study we utilized a feeder layer of 3T3 cells to grow homogeneous cultures of human ectocervical epithelial cells and demonstrated the presence of the cornified envelope precursor, involucrin. Treatment of these cultures with 1 nM Ro 13-6298, a synthetic analogue of trans-retinoic acid, suppresses envelope formation 6-fold with half-maximal suppression at 0.005-0.01 nM. Treatment with 1 microM hydrocortisone elevates envelope production 2.5-fold. Sex steroids also regulate desquamation: 10 nM diethylstilbestrol, a synthetic estrogen, increases envelope levels 2- to 3-fold, while 300 nM progesterone reduces envelope production 2- to 3-fold. In spite of the retinoid-, glucocorticoid- and sex-steroid-stimulated changes in envelope production, the level of the envelope precursor, involucrin, remains constant. Our results suggest: (1) that, in vivo, ectocervical cell squame formation is regulated by the combined direct action of estrogens, progestins, glucocorticoids and retinoids; and (2) that envelope formation is not regulated by changes in the cellular content of the envelope precursor, involucrin. We present a model summarizing the estrogen, progestin, glucocorticoid and retinoid effects on ectocervical epithelial cell function.

Preliminary experience with in vitro fertilization-surrogate gestational pregnancy.

This report documents the medical details and 3-year follow-up evaluation of the infertile and surrogate couples involved in the first successful in vitro fertilization gestational surrogate pregnancy and summarizes clinical experience and outcome of all patients treated to date. Results of the first 28 couples treated are presented. The pregnancy rate for 39 cycles reaching attempts at oocyte retrieval is 18%. The procedural aspects, ethical issues, legal issues, and subsequent program development are summarized. Recommendations are of a similar program. There are numerous potential pitfalls and traps for the unwary, but our experience has thus far been gratifyingly positive, and we endorse the further provision, observation, and documentation of this controversial approach to the care of the infertile couple.

In vitro fertilization (IVF)-surrogacy: application of IVF to women without functional uteri.

Women with absent or dysfunctional uteri consented to controlled ovarian stimulation, ovum aspiration, in vitro fertilization, and embryo culture. Cleaving preembryos were transferred to recipient (surrogate) women whose menstrual cycles were in approximate synchrony with the ovum donor. None of the embryo recipients received medication. Six cases are described, resulting in one spontaneous loss at 6 weeks, four full-term deliveries, and one ongoing pregnancy. HLA typing demonstrated all babies to be genotypic offspring of the gamete donors.

Implementation of an in vitro fertilization program.

The establishment of an in vitro fertilization program is described. Organization of the physical laboratory, media formulation, and preliminary mouse embryo culture are discussed. Parameters of patient eligibility, ovarian stimulation, and laparoscopy are also defined. At the conclusion of Phase I, 17 patients were induced; 10 went to laparoscopy, and at least one four-cell embryo was returned to 7, resulting in one continuing pregnancy.

The roles of NADH in the support of steroid aromatization by human placental microsomes.

The ability of NADH to function as an alternative cofactor for the support of estrogen biosynthesis was validated. NADH supported rates of aromatization of up to 80% of those obtained with NADPH, with an apparent Km of 0.70 mM, and stimulated the NADPH-supported reaction only when supplies of the normal cofactor were limiting, both additive and synergistic effects being observed. NADH-supported aromatization was inhibited competitively by NADP+ and 2'-AMP with Ki values of 5 microM and 22 microM, respectively. Support by both cofactors was lost in parallel with the selective removal of NADPH-cytochrome c reductase from microsomes by graded subtilisin treatment. NADH-supported aromatization was differentiated from NADPH-supported aromatization by its sensitivity to inhibition by NAD+ and its response to changes in ionic strength. NADH appears to function, at high concentrations, as a surrogate for NADPH at the reduced nucleotide-binding site of NADPH-cytochrome c reductase but additional roles for NADH are also suggested both when acting alone and as a supplement to NADPH. A common oxidase (cytochrome P-450) appears to catalyze both NADH- and NADPH-supported aromatization.

Inhibitor specificity of the placental microsomal oxidase system responsible for the aromatization of epitestosterone (17 alpha-hydroxy-4-androsten-3-one).

Human placental microsomes converted epitestosterone to estradiol-17 alpha at rates of 23-48 pmol/min X mg protein with a Km of 113 microM. Activity was inhibited 70-90% by concentrations of CO, metyrapone, n-octylamine, 7,8-benzoflavone and 7-ethoxycoumarin which had no effect on the aromatization of 4-androstene-3, 17-dione. Conversely, cyanide and azide were more effective inhibitors of the conversion of the latter androgen. A variety of neutral steroids inhibited the aromatization of epitestosterone with 19-norsteroids being particularly effective, but competitive effects could not be demonstrated. Both 17 beta-hydroxy-4-estren-3-one and 16 alpha-hydroxy-4-androstene-3,17-dione caused a mixed inhibition. A number of phenolic steroids were also inhibitory with 16-oxo compounds being particularly effective. Inhibition by estrone was non-competitive (Ki = 16 microM). The aromatization of epitestosterone resembles placental microsomal oxidase activities against estrone and benzo [a]pyrene in its inhibitor specificity and epitestosterone may be the native substrate for an oxidase also active in the metabolism of aromatic xenobiotic chemicals.